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目的构建携带FasL的非增殖腺病毒载体,并研究其生物学特性。方法将人FasL全长cDNA克隆到非增殖腺病毒载体pSuCMV中,获得pSuCMVFasL重组子,经293包装细胞包装,获得氨苄青霉素抗性克隆,命名为pSuCMVFasL。体外感染人近端肾小管上皮细胞株HK-2细胞72h后,Western blot检测其在HK-2细胞中的表达。结果获得携带FasL非增殖腺病毒,滴度为7.3×109pfu/ml;Western blot法检测在其感染的HK-2细胞中能检测到重组的蛋白表达。结论将FasL基因导入非增殖腺病毒载体是一种行之有效的基因转染途径,制备的非增殖腺病毒在体外能有效感染HK-2细胞,为进行体内移植免疫耐受的基因治疗研究奠定基础。
Objective To construct a non-proliferating adenovirus vector carrying FasL and investigate its biological characteristics. Methods The full-length human FasL cDNA was cloned into the non-proliferating adenovirus vector pSuCMV to obtain the recombinant pSuCMVFasL. The recombinant plasmid pCMVFasL was packaged in 293 packaging cells to obtain the ampicillin-resistant clone named pSuCMVFasL. The human proximal tubular epithelial cell line HK-2 cells were infected with in vitro for 72 h, and the expression of HK-2 in HK-2 cells was detected by Western blot. Results The recombinant adenovirus carrying FasL was obtained with a titer of 7.3 × 109 pfu / ml. The recombinant protein was detected by Western blot in transfected HK-2 cells. Conclusion The introduction of FasL gene into non-proliferating adenovirus vector is an effective way of gene transfection. The prepared non-proliferating adenovirus can effectively infect HK-2 cells in vitro and lay the foundation for the gene therapy research of in vivo transplantation tolerance basis.